Method for discrimination of metaplasias from neoplastic or preneoplastic lesions

ABSTRACT

The present invention relates to a method for discrimination of p16INK4a overexpressing metaplasias from neoplastic or preneoplastic p16INK4a overexpressing lesions by determination of the level of high risk HPV encoded gene-products such as e.g. HPV E2 and/or HPV E7 molecules in biological samples in the course of cytological testing procedures. The method thus enables for reduction of false positive results in the p16INK4a based detection of anogenital lesions in cytological testing procedures.

This application is a continuation of U.S. application Ser. No.14/325,282, filed Jul. 7, 2014; which is a continuation of U.S.application Ser. No. 13/137,547, filed Aug. 25, 2011, now U.S. Pat. No.8,815,520; which is a continuation of U.S. application Ser. No.12/164,934, filed Jun. 30, 2008, now U.S. Pat. No. 8,043,819; which is acontinuation of U.S. application Ser. No. 10/511,108, filed Oct. 8,2004, now U.S. Pat. No. 7,422,859; which is a National Stage ofInternational Application PCT/EP2003/050096, filed Apr. 8, 2003,published Jan. 15, 2004, under PCT Article 21(2) in English; whichclaims the priority of European Application No. 02007954.7, filed Apr.9, 2002. The contents of the above-identified applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates to a method for discrimination ofp16^(INK4a) overexpressing metaplasias from neoplastic or preneoplasticp16^(INK4a) overexpressing lesions by determination of the level of highrisk HPV encoded gene-products such as e.g. HPV E2 or E7 molecules inbiological samples in the course of cytological testing procedures. Themethod thus enables for reduction of false positive results in thep16^(INK4a) based detection of anogenital lesions in cytological testingprocedures.

BACKGROUND OF THE INVENTION

The detection of the overexpression of p16^(INK4a) in biological sampleshas proven as a useful marker in the detection of anogenital lesionssuch as carcinoma of the uterine cervix (see WO00/01845; Klaes et al.,Int. J. Cancer: 92, 276-284 (2001)). The method based onp16^(INK4a)-specific immuno-chemical staining allows for a sensitive andspecific identification of dysplastic cells in tissue section and incytological samples.

In immuno-histochemical examinations of tissues, neoplastic cells can bestained using a p16^(INK4a) specific antibody mediated stainingprocedure. The histological diagnosis of neoplastic lesions can thus besupported by a staining based on a molecular marker characteristic fortransformation of cells in anogenital lesions. The diagnosis, whether ornot cells are neoplastic, in these procedures is not solely based on thep16^(INK4a) specific staining, but does also rely on the histologicalinformation.

This is due to the fact, that in about 30% of samples, metaplastic cellsshow some immunoreactivity with p16^(INK4a) specific antibodies, andthus are stained in the course of the procedures. Yet the stainingpattern yielded from these metaplastic cells differs from the pattern,which renders from neoplastic lesions. Metaplastic cells give rise to apatchy or focal staining pattern, whereas neoplastic lesions give riseto diffuse staining pattern. Moreover, the staining intensities ofmetaplastic cells are predominantly less than that of neoplastic cells.

The common methods used in screening tests for the early detection ofneoplasias do not employ histology based tests, but do rather rely oncytological testing procedures. Yet especially in cases, when there isno histological information available concerning the architecture oftissues, such as for example in cytological examinations, testing forp16^(INK4a) overexpression alone may lead to false positive results.This is due to the fact, that the metaplastic cells expressingp16^(INK4a) at detectably elevated levels, may not be differentiated bymeans of a histological staining patterns.

The percentage of cells showing overexpression of p16^(INK4a) increasesin the course of emergence of dysplasias. So in neoplastic orpreneoplastic stages, when only a restricted population of neoplastic orpreneoplastic cells is present in samples the immunoreactivity ofp16^(INK4a) may be weak. This weak immunoreactivity may be of about thelevel as the level caused by metaplastic cells. In later stages ofdysplasias the overall immunoreactivity of p16^(INK4a) is stronger andso neoplastic lesions are easily discernible from metaplasias even in acytological testing format. This might lead to cases, where the presenceof metaplastic cells expressing p16^(INK4a) might be confused with thepresence of neoplastic cells, and thus produces a false positive result.

Especially in screening tests, where the detection of early stages ofneoplasias is desirable this condition is quite unpleasant. This isespecially true, as the p16^(INK4a) based diagnosis has proven to be avaluable tool in histological examinations and the application incytological based screening procedures would be able to enhance theseestablished procedures.

To reduce false positive results in cytological testing formats and soto further enhance the fidelity of the p16^(INK4a) mediated diagnosis ofanogenital lesions, a method for discriminating the metaplasias fromneoplastic and dysplastic lesions would be desirable. The problem in theart especially pertains to early stages of neoplasias, when thepercentage of cells showing p16^(INK4a) overexpression is still at alevel, that might be confused with levels of normally occurringp16^(INK4a) overexpressing proliferating metaplastic cells. Thus, usefulmeans for solving the present problem have to involve parameters, thatcharacterize early stages of neoplasias of the anogenital tract. Anycharacteristics of dysplasias and/or neoplasias emerging during theprogress of tumorigenesis, thus proving as diagnostic tools for highgrade dysplasias, and are limited in early stages of tumorigenesis arenot suitable for the method according to the present invention.

SUMMARY OF THE INVENTION

A method for the discrimination of metaplasias from neoplastic andpreneoplastic lesions is provided within the embodiments claimedaccording to the present invention.

For supporting the discrimination of metaplasias from neoplastic lesionsin testing procedures based on the overexpression of p16^(INK4a), amarker molecule that is expressed in neoplastic and/or preneoplasticcells and tissues and is not expressed in metaplastic cells would besuitable.

E2 of HPV is especially expressed in lower grade CIN lesions and theexpression decreases with ascending CIN grades (Stevenson et al., J.Gen. Virol., 81, 1825-32 (2000)). In most invasive carcinomas, noexpression of E2 protein is detectable. This may be due to the fact,that parts of the E2 gene are lost during integration of the HPV DNA,into the host genome. Thus in persistent HPV infections havingintegrated HPV DNA no or low E2 expression may be detected.

Due to these facts E2 protein proves to be a marker for early stages oflesions associated with high risk HPV infections. In contrast,p16^(INK4a) is a marker, which is overexpressed even in early stages ofanogenital lesions, the expression level of which increases in thecourse of progression of dysplastic lesions. The fact that E2 isespecially expressed in early stages of neoplasias makes it particularlyuseful for early detection methods. The high expression level of E2protein in early stages of HPV infection allows to identify infectedcells, before there is an abundant number of copies of the virus presentin the tested cells.

The L1 and L2 gene-products are also useful for the method according tothe present invention due to their high expression level predominantlyin early stages of viral infection before integration has occurred. Theexpression of these gene-products is also reduced in persistentinfection of HPV.

The inventors now have found, that cells expressing high risk HPVgene-products such as HPV E2 may serve to discriminate early neoplasticor dysplastic lesions detectable by p16^(INK4a)-specific immuno-chemicalstaining from metaplasias, which may also comprise cells immunoreactivewith p16^(INK4a), in the course of cytological testing procedures.

Cells expressing other HPV encoded gene-products, that are detectable onan expression level as mRNA or polypeptide in neoplastic stages orpreneoplastic stages, may also serve for the discrimination ofneoplastic and/or preneoplastic lesions from metaplasias overexpressingp16^(INK4a) according to the present invention. Examples of such HPVencoded gene-products include HPV E6, E7, L1 or L2 proteins or mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows metaplastic cells immunochemically stained with an antibodyspecific for p16^(INK4a).

For experimental details see Example 1; the cells clearly react with theantibodies against p16^(INK4a).

FIG. 2 shows metaplastic cells immunochemically stained with an antibodyspecific for HPV E2.

For experimental details see Example 1; the cells do not showimmunoreactivity with the polyclonal antibodies directed against HPV E2protein.

FIG. 3 shows metaplastic cells immunochemically stained with an antibodyspecific for HPV L1.

For experimental details see Example 1; the cells do not showimmunoreactivity with the polyclonal antibodies directed against HPV L1protein.

FIG. 4 shows dysplastic cells immunochemically stained with an antibodyspecific for p16^(INK4a). For experimental details see Example 1; thecells clearly react with the antibodies against p16^(INK4a).

FIG. 5 shows dysplastic cells immunochemically stained with an antibodyspecific for HPV E2. For experimental details see Example 1; the cellsshow clear immunoreactivity with the polyclonal antibodies directedagainst HPV E2 protein.

FIG. 6 shows dysplastic cells immunochemically stained with an antibodyspecific for HPV L1. For experimental details see Example 1; the cellsshow clear immunoreactivity with the polyclonal antibodies directedagainst HPV L1 protein.

FIG. 7 shows metaplastic cells immunochemically stained with an antibodyspecific for p16^(INK4a). For experimental details see Example 3; thecells clearly react with the antibodies against p16^(INK4a).

FIG. 8 shows metaplastic cells immunochemically stained with an antibodyspecific for HPV E7. For experimental details see Example 3; the cellsdo not show immunoreactivity with the monoclonal antibodies directedagainst HPV E7 protein.

FIG. 9 shows dysplastic cells immunochemically stained with an antibodyspecific for p16^(INK4a). For experimental details see Example 3; thecells clearly react with the antibodies against p16^(INK4a).

FIG. 10 shows dysplastic cells immunochemically stained with an antibodyspecific for HPV E7. For experimental details see Example 3; the cellsclearly react with the antibodies against HPV E7 protein.

FIG. 11 shows dysplastic cells immunochemically double stained withantibodies specific for HPV E7 and antibodies specific for p16^(INK4a).For experimental details see Example 3; the cells clearly react with theantibodies against both HPV E7 and p16^(INK4a) proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for discrimination ofneoplastic, preneoplastic and/or dysplastic lesions from metaplasiascomprising p16^(INK4a) overexpressing cells, in biological samples in acytological testing procedure based on the detection of the presence orabsence of cells expressing high risk HPV gene-products in saidbiological samples. HPV gene-products useful for the method disclosedherein are gene-products, which are highly expressed especially in earlystages of neoplastic and preneoplastic lesions. In one embodiment of theinvention HPV E2 protein or mRNA may serve as a marker fordiscrimination of metaplasias from early neoplastic or preneoplasticlesions in samples. Furthermore HPV E6, E7, L1 or L2 protein and/or mRNAalso proved to be suitable for performing the discrimination accordingto the invention disclosed herein.

Discrimination as used in the context of the present invention shallcomprise an assessment whether a sample is to be classified in one oranother way. In a preferred embodiment of the invention thediscrimination pertains to the assessment of a tissue or componentsthereof being neoplastic or being metaplastic. Thus the discriminationas used herein is a judgement about the growth properties of cells in asample.

The discrimination according to the present invention is based on thepresence or absence of cells expressing a high risk HPV gene-product andon the presence or absence of cells overexpressing p16^(INK4a) in saidsample. The cells expressing the high risk HPV gene-products such as E2need not be the same cells as those overexpressing p16^(INK4a) althoughthe expression of both marker molecules may occur in the same cells.

Thus the presence of cells expressing E2 gene-products in a samplesimultaneously with the presence of cells overexpressing p16^(INK4a)(other cells or the same cells coexpressing both markers) according tothe present invention serves to discriminate neoplastic or preneoplasticlesions from metaplasias.

HPV encoded gene-products as used in the context of the presentinvention shall be any mRNA transcribed from a gene of the HPV genome orany polypeptide translated from such an mRNA. HPV gene-products suitablefor the method according to the present invention are gene-productsencoded by the E6, E7, L1 and L2 genes. In one especially preferredembodiment of the present invention the HPV gene-product is encoded bythe HPV E2 gene.

HPV herein means human papilloma virus. HPV as used herein shallcomprise any high risk subtype of HPV. In a preferred embodiment of thepresent invention the HPV subtype is a cancer associated HPV subtypesuch as e.g. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56 and 58. In anespecially preferred embodiment the HPV high risk subtypes are HPV 16,18, 39 or HPV 45. Subtyping of the HPV shall comprise any methodsuitable of the determination of the particular HPV subtype present in abiological sample.

The method for detection of the level of the HPV encoded gene-productsaccording to the present invention is any method, which is suited todetect very small amounts of specific biological molecules in biologicalsamples. The detection reaction according to the present invention is adetection either on the level of nucleic acids or on the level ofpolypeptides.

The HPV gene-products may be detected using reagents that specificallyrecognise these molecules. The detection reaction for the HPVgene-products may comprise one or more reactions with detecting agentseither recognizing the initial marker molecules or recognizing the priormolecules used to recognize other molecules.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the HPVgene-products. The reporter reaction may be for example a reactionproducing a coloured compound, a bioluminescence reaction, afluorescence reaction, generally a radiation emitting reaction etc.

In a preferred embodiment, different marker molecules may be recognizedby agents, that produce different reporter signals, so that the signalsreferring to marker molecules could be distinguished. In one preferredembodiment of the invention the detection of the expression of high riskHPV gene-products is carried out simultaneously with the detection ofthe overexpression of p16^(INK4a) In this case the reporter reaction mayfor example employ different fluorescent labels for the differentmolecules detected.

Applicable formats for the detection reaction according to the presentinvention may be, blotting techniques, such as Western-Blot,Southern-blot, Northern-blot. The blotting techniques are known to thoseof ordinary skill in the art and may be performed for example aselectro-blots, semidry-blots, vacuum-blots or dot-blots. Amplificationreaction may also be applicable for the detection of e.g. nucleic acidmolecules.

In one preferred embodiment of the invention the detection of the levelof HPV gene-products is carried out by detection of the respective mRNAor fragments thereof present in the sample. The means for detection ofnucleic acid molecules are known to those skilled in the art. Theprocedure for the detection of nucleic acids can for example be carriedout by a binding reaction of the molecule to be detected tocomplementary nucleic acid probes, proteins with binding specificity forthe nucleic acids or any other entities specifically recognizing andbinding to said nucleic acids.

This method can be performed as well in vitro as directly in situ forexample in the course of a detecting staining reaction. Another way ofdetecting the HPV mRNAs in a sample performed in the method according tothe present invention is an amplification reaction of nucleic acids,which can be carried out in a quantitative manner such as for examplethe polymerase chain reaction. In a preferred embodiment of the presentinvention, real time RT PCR is used to quantify the level of HPV mRNA insamples of tumors.

In another preferred embodiment of the invention, the detection of thelevel of HPV gene-products is carried out by determining the level ofexpression of a protein. The determination of the HPV gene-product onthe protein level can, for example, be carried out in a reactioncomprising a binding agent specific for the detection of the particularHPV polypeptide.

The binding agents can be used in many different detection techniquesfor example in western-blot, ELISA or immuno-precipitation. Generallypolypeptide binding agent based detection can be carried out as well invitro as directly in situ for example in the course of animmuno-histochemical staining reaction. Any other method for determiningthe amount of particular polypeptides in biological samples can be usedaccording to the present invention.

Binding agents as used in the context of the present invention for thedetection of the level of either HPV polypeptides or p16^(INK4a)polypeptides may comprise antibodies and antigen-binding fragments,bifunctional hybrid antibodies, peptidomimetics containing minimalantigen-binding epitopes etc.

An antibody or antigen-binding agent is said to react specifically, ifit reacts at a detectable level with a protein disclosed herein, anddoes not significantly react with other proteins. The antibodiesaccording to the present invention may be monoclonal or polyclonalantibodies. As used herein, the term antibody or monoclonal antibody ismeant to include intact molecules as well as antibody fragments.Moreover, antibodies of the present invention include chimeric, singlechain, and humanized antibodies.

According to the present invention binding agents may be used isolatedor in combination. By means of combination it is possible to achieve ahigher degree of sensitivity. The term antibody, preferably, relates toantibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations.

Monoclonal antibodies are made from antigen containing fragments of thepolypeptide of the invention using any of a variety of techniques knownto those of ordinary skill in the art; see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, 1988.In one such technique, an immunogen comprising the antigenic polypeptideor a synthetic part thereof is initially injected into any of a widevariety of mammals (e.g., mice, rats, rabbits, sheep and goats). In thisstep, the polypeptides of this invention may serve as the immunogenwithout modification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

The methods used for the detection of the presence or absence ofoverexpression of p16^(INK4a) according to the present invention are thesame methods mentioned above for the detection of HPV gene-products.

The HPV gene-products may according to the present invention be detectedsimultaneously with the presence or absence of the overexpression ofp16^(INK4a). In this context simultaneously according to the presentinvention shall mean either literally at the same instant or within thesame testing procedure, whereby the single detection steps aretemporarily consecutive.

A sample according to the method of the present invention may compriseany sample comprising cells of anogenital origin. Samples may comprisee.g. secretions, smears, body fluids, and cell-samples.

In one embodiment of the present invention, samples comprise cells ofthe uterine cervix. In a preferred embodiment of the present inventionthe sample of cervical cells may be prepared according to a classicalPap smear. In a further preferred embodiment of the present invention,the sample may be prepared as a monolayer or thin layer preparation ofthe cytological specimen.

Preparation of a sample may comprise e.g. obtaining a sample of atissue, of a body fluid, of cells from a patient. According to thepresent invention, preparation of the sample may also comprise severalsteps of further preparations of the sample, such as preparation ofdissections, spreading or applying the cells to be examined ontomicroscopic slides, preparation of tissue arrays, isolation ofpolypeptides or nucleic acids, preparation of solid phase fixed peptidesor nucleic acids or preparation of beads, membranes or slides to whichthe molecules to be determined are coupled covalently or non-covalently.

The neoplastic lesions to which the method according to the presentinvention may be applied comprise any anogenital lesion, which ischaracterized by the overexpression of p16^(INK4a), which furthermoreshows expression of HPV gene-products. In one preferred embodiment ofthe present invention the anogenital lesion is a lesion of the uterinecervix.

Another aspect of the present invention is a testing kit for performingthe method according to the present invention. The kit may be forexample a diagnostic kit or a research kit.

A kit according to the present invention comprises at least an agentsuitable for detecting the HPV gene-products and an agent suitable forthe detection of the presence or absence of the overexpression ofp16^(INK4a).

Thus a kit according to present invention may comprise:

a) reagents for the detection of the HPV gene-products

b) reagents for the detection of the p16^(INK4a) overexpression

c) reagents and buffers commonly used for carrying out the detectionreaction, such as buffers, detection-markers, carrier substances andothers

d) a p16^(INK4a) sample for carrying out a positive control reaction

e) a HPV gene-product sample for carrying out a positive controlreaction

The reagents for the detection of the HPV gene-products and/orp16^(INK4a) may include any agent capable of binding to the HPVgene-products and/or p16^(INK4a) molecule. Such reagents may includeproteins, polypeptides, nucleic acids, peptide nucleic acids,glycoproteins, proteoglycans, polysaccharides or lipids.

The HPV gene-product and/or p16^(INK4a) sample for carrying out apositive control may comprise for example nucleic acids in applicableform, such as solution or salt, peptides in applicable form, tissuesection samples or positive cells.

In a preferred embodiment of the invention, the detection of the HPVgene-products and/or p16^(INK4a) is carried out on the level ofpolypeptides. In this embodiment, the binding agents may be for exampleantibodies specific for the HPV gene-products or p16^(INK4a) orfragments thereof.

In an other embodiment of the test kit, the detection of the HPVgene-products and/or p16^(INK4a) is carried out on the nucleic acidlevel. In this embodiment of the invention, the reagent for thedetection may be, for example, a nucleic acid probe or a primerreverse-complementary to said HPV gene-product and/or p16^(INK4a)nucleic acids.

The present invention provides a method for the discrimination ofneoplastic and preneoplastic anogenital lesion identifiable byassessment of the overexpression of p16^(INK4a) from metaplastic cells,which detectably express p16^(INK4a) in the course of cytologicaltesting procedures. The method is based on the detection of expressedgene-products of high risk HPV. It turned out, that high risk HPVgene-products expressed in high levels in the early stages of neoplasiasand in preneoplasias are suitable for this discrimination. This is dueto the fact that the percentage of cells in a biological sample in earlystages of neoplasias overexpressing p16^(INK4a) renders a level ofp16^(INK4a) molecules, that there remains the possibility, that thelevel recurs to metaplastic rather than neoplastic cells. Thus theproblem to be solved was to provide a method for discrimination betweenneoplastic and metaplastic cells especially in early stages ofneoplasias, when cytological diagnostic methods based on the p16^(INK4a)overexpression needs a further information for the identification ofmetaplastic cells.

Furthermore the present invention provides a kit for performing themethod according to the present invention.

EXAMPLES

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein.

Example 1: Immunochemical Detection of the Expression of HPV E2, L1 andp16^(INK4a) in Samples of the Uterine Cervix

Smears of the cervix uteri were immunocytochemically stained usingantibodies specific for p16^(INK4a), polyclonal antibodies specific forHPV L1 and polyclonal antibodies specific for HPV E2 protein.

For rehydration, the spray-fixed smears prepared on slides wereincubated in fresh 50% EtOH on a rocking device. The PEG film producedby the fixation procedure was removed by intensive rinsing. The smearswere rinsed in aqua bidest. Antigen Retrieval was carried out with 10 mMcitrate buffer (pH 6.0). Then the slides were heated in a water bath for40 min at 95° C., cooled down to RT for 20 minutes, transferred towashing buffer (PBS/0.1% Tween20) and finally surrounded with alipid-pencil.

For inactivation of endogenous peroxidase, the samples were incubatedwith 3% H₂O₂ for 20 min at RT and afterwards washed in PBS/0.1% Tween20for 5 min. The proteinblock was carried out with horse-serum(Vectastain®-Kit) (Dilute 1:50 with PBS/0.1% Tween20). The smears wereincubate for 20 min at RT and then rinsed off carefully. Then blockingof non-specific binding of avidin—reagent was performed as follows:Samples were incubated with avidin blocking solution(ready-to-use/Vector) for 15 min at RT and then washed carefully withpipette. For blocking of non-specific binding of biotin-reagent thesmears were incubated with biotin blocking solution(ready-to-use/Vector) for 15 min at RT and then rinsed off carefully.

Then follows incubation with a p16^(INK4a) specific primary antibody orpolyclonal antibodies directed against HPV E2 protein or polyclonalantibodies raised against high risk (HPV16) HPV L1 protein; the sampleswere incubated for 60 min at RT, washed in PBS/0.1% Tween20 for 5 min(two times) and afterwards incubated with a biotinylated SecondaryAntibody (horse-anti-mouse-IgG) (Vectastain®-Kit/Dilute 1:200 inPBS/0.1% Tween20+Horse-Serum) for 30 min at RT and washed in PBS/0.1%Tween20 for 5 min (two times). Following an incubation with AB-Complex(Avidin-Biotin-HRP) (Vectastain®-Kit/Dilute 1:50 in PBS/0.1% Tween20)was performed for 30 min at RT followed by washing steps in PBS/0.1%Tween20 for 5 min (two times).

Signal detection was carried out with Substrate-Chromogen-Complex(H₂O₂/AEC) as follows: First the samples were incubate for 30 min at RTwith the substrate-chromogen complex, then the reaction was stopped inaqua bidest. Finally, a counterstain with Mayers Hematoxylin wasperformed and the slides were mounted with Glycerin gelatin.

The microscopic examination of the slides revealed that cellsimmunoreactive with p16^(INK4a) antibody together with cellsimmunoreactive with HPV E2 protein antibody can only be found in samplesthat were microscopically identified as samples of neoplastic lesions.Cells stained by the p16^(INK4a) specific reaction, which wereoriginating from metaplasias, were not stained by the reaction specificfor the HPV E2 protein. The microscopic inspection of the HPV L1 stainedslides showed that metaplastic cells were not immunoreactive with theantibodies directed against HPV L1 protein. Samples containingdysplastic cells in contrast comprise cells immunoreactive with HPV L1antibody and cells immunoreactive with p16^(INK4a) antibody. So incontrast to dysplasias, no cells in metaplasias were stained using theHPV L1 specific antibody.

The results show that the staining with reagents specific for HPV E2 orL1 allowed to discriminate p16^(INK4a) overexpressing metaplasias fromdysplasias.

Example 2: Detection of Cells Expressing HPV E2, HPV L1 or p16^(INK4a)in Samples of the Uterine Cervix by in Situ Hybridization

Smears of the uterine cervix are semi-quantitatively analysed for themRNA level of p16^(INK4a) and HPV E2 and L1 in an in-situ stainingreaction. The staining reaction is performed as follows:

For rehydration, the spray-fixed smears prepared on slides are incubatedin fresh 50% EtOH on a rocking device. The PEG film produced by thefixation procedure is removed by intensive rinsing. Then the smears arerinsed in aqua bidest. The smears are incubated with proteinase K (10μg/ml in PBS) for 10 min at 37° C. Then the slides are transferred towashing buffer (PBS/0.1% Tween20) and finally surrounded with alipid-pencil.

The hybridization mixture is prepared by mixing 50 μl of ready to usehybridization buffer (DAKO A/S, Glostrup, Denmark) with about 5-10 pmolof the probes. The probes are fluorescein-labelled oligonucleotides ofsequences complementary to the respective mRNAs.

The hybridization mixture is heated to 95° C. and afterwardsequilibrated to 37° C. After the boiling procedure, the smears areincubated with each 50 μl of the hybridization mixture for 4 hours at42° C. The samples are washed in excess volumes of the wash buffers twotimes in 2×SSC at 37° C. for 15 min and once in 1×SSC at 37° C. for 15min. Then the smears are rinsed two times at room temperature in 2×SSC.Following this washing procedure, the dissections are incubated for 30min with blocking buffer (NEN, Blockingpugger) at room temperature,followed by 1 hour incubation with a 1:100 diluted (in Blocking buffer,see above) anti-Fluorescein-alkaline phosphatase (DAKO A/S). The smearsare then washed 2 times in 1×PBS/0.1% Triton X-100 for 10 min at roomtemperature, followed by one wash step with 1×PBS, 50 mM MgCl₂ (pH 9.2)for 10 min at room temperature. Then the staining reaction is performedwith NBT/BCIP (Sigma) for 30 min to 2 hours at room temperature. Thestaining reaction is stopped by a short incubation with 1 mM EDTA inPBS. Finally the smears are dipped in H₂O_(dest.) and embedded withAquaTex (Merck). Then the stained dissections can be analysedmicroscopically.

Microscopic analysis reveals that metaplasias comprise cells expressingp16^(INK4a) but no cells expressing HPV L1 or E2 mRNA. In dysplasticlesions of the cervix uteri, cells expressing p16^(INK4a) and cellsexpressing HPV L1 and HPV E2 mRNAs can be found.

This result indicates that the method according to the present inventionmay be used for the discrimination of metaplasias and neoplasticlesions.

Example 3: Immunocytochemical Detection of the Expression of HPV E7, andp16^(INK4a) in Samples of the Uterine Cervix

ThinPrep® thin layers of smears of the cervix uteri wereimmunocytochemically stained using antibodies specific for p16^(INK4a)and monoclonal antibodies specific for HPV E7 protein.

For rehydration, the spray-fixed smears prepared on slides wereincubated in fresh 50% EtOH on a rocking device. The PEG film producedby the fixation procedure was removed by intensive rinsing. Then thesmears were rinsed in aqua bidest. Antigen Retrieval was carried outwith 10 mM citrate buffer (pH 6.0). Thereafter, the slides were heatedin a water bath for 40 min at 95-98° C., cooled down to RT for 20minutes, transferred to washing buffer (Dakocytomation, S3006) andfinally surrounded with a lipid-pencil.

For inactivation of endogenous peroxidase, the samples were incubatedwith 3% H₂O₂ (Dakocytomation; S2023) for 5 min at RT and afterwardswashed in wash buffer (Dakocytomation, S3006) for 5 min, then followedby incubation with a p16^(INK4a) specific primary antibody or monoclonalantibodies directed against high risk (HPV16) E7 protein. The sampleswere incubated for 30 min at RT, washed in wash buffer for 5 min andafterwards incubated with the EnVision system (Dakocytomation, K4001),consisting of goat anti-mouse antibodies conjugated to a dextran polymerand peroxidase enzymes, for 30 min at RT and washed in wash buffer for 5min (three times).

Signal detection was carried out with Substrate-Chromogen-Complex(diaminobenzidine (DAB+), Dakocytomation, K3468) by incubation for 10min at RT. Then the reaction was stopped in aqua dest. Finally acounterstain with Mayers Hematoxylin was performed and the slides weremounted with Faramount (DakoCytomation, S3025).

The microscopic examination of the slides revealed, that cellsimmunoreactive with p16^(INK4a) antibody together with cellsimmunoreactive with HPV E7 protein antibody can only be found insamples, that were microscopically identified as samples of neoplasticlesions. Cells stained by the p16^(INK4a) specific reaction, which wereoriginating from metaplasias, were not stained by the reaction specificfor the HPV E7 protein.

To validate the results given herein, a double staining of cytologicalspecimens was performed using a FITC-labelled HPV E7 antibody (antibodyas given above) in combination with the p16^(INK4a) antibody as usedabove. The staining was performed as given above adapted to a doublestaining procedure. The necessary variations are known to those of skillin the art.

In double stained specimens, no double stained metaplastic cells werefound; in contrast dysplastic cells showed double staining forp16^(INK4a) and HPV E7 protein.

The results show that the staining with reagents specific for HPV E7allows to discriminate p16^(INK4a) overexpressing metaplasias fromdysplasias.

What is claimed is:
 1. A method for screening a uterine cervicalcytology sample, comprising: (a) obtaining at least one slide having theuterine cervical cytology sample deposited thereon; (b) staining theuterine cytology sample with a labeled oligonucleotide probe thatspecifically binds p16^(INK4a) mRNA and is complementary to p16^(INK4a)mRNA in the sample by in situ hybridization, wherein the label of theoligonucleotide probe that is complementary to the p16^(INK4a) mRNAgenerates a first reporter signal; (c) staining the uterine cervicalcytology sample with a labelled oligonucleotide probe that specificallybinds a high risk HPV (hr-HPV) gene-product and is complementary to amRNA of the high risk HPV (hr-HPV) gene-product by in situhybridization, wherein said high risk HPV gene-product is encoded by agene selected from the group consisting of HPV E7 gene, HPV E2 gene, HPVE6 gene, HPV L1 gene, and HPV L2 gene, wherein the label of theoligonucleotide probe that is complementary to the mRNA of the hr-HPVgene product generates a second reporter signal; and (d) detecting inthe uterine cytology sample: (d1) the simultaneous-presence of cellsstained with the label generating the first reporter signal and cellsstained with the label generating the second reporter signal, or (d2)the presence of cells stained with the label generating the firstreporter signal and the absence of cells stained with the labelgenerating the second reporter signal.
 2. The method according to claim1, wherein the high risk HPV gene-product is encoded by the HPV E7 gene.3. The method according to claim 1, wherein the high risk HPVgene-product is encoded by the HPV E2 or the HPV E6 gene.
 4. The methodaccording to claim 1, wherein the high risk HPV gene-product is encodedby the HPV L1 or the HPV L2 gene.
 5. The method of claim 1, wherein thestaining of (b) and the staining of (c) are performed on the same slide,and wherein the first reporter signal and the second reporter signal aredifferent.
 6. The method of claim 5, wherein the agent generating thefirst reporter signal and the agent generating the second reportersignal are different fluorescent labels.
 7. The method of claim 5,wherein the agent generating the first reporter signal and the agentgenerating the second reporter signal are different colored compounds.8. The method of claim 1, wherein the staining of (b) and the stainingof (c) are performed on different slides.
 9. The method of claim 8,wherein the agent generating the first reporter signal and the agentgenerating the second reporter signal are fluorescent labels or coloredcompounds.
 10. The method according to claim 9, wherein the coloredcompound that generates the first reporter signal and the secondreporter signal is diaminobenzidine.
 11. A method of preparing a uterinecervical cytology sample for microscopic evaluation, said methodcomprising: (a) obtaining a slide having the uterine cervical cytologysample deposited thereon; (b) staining the uterine cervical cytologysample on the slide by in situ hybridization for both: (b1) p16^(INK4a)mRNA, wherein the stain for p16^(INK4a) mRNA is an agent thatspecifically binds p16^(INK4a) mRNA and gene rates a first reportersignal; and (b2) a high risk HPV (hrHPV) mRNA encoded by a gene selectedfrom the group consisting of HPV E7 gene, HPV E2 gene, HPV E6 gene, HPVL1 gene, and HPV L2 gene, wherein the stain for the hrHPV is an agentthat specifically binds the high risk HPV mRNA and generates a secondreporter signal, wherein the first reporter signal is distinguishablefrom the second reporter signal.
 12. The method of claim 11, wherein theagent generating the first reporter signal and the agent generating thesecond reporter signal are different fluorescent labels or differentcolored compounds.
 13. The method according to claim 11, wherein thehigh risk HPV gene-product is encoded by the HPV E7 gene.
 14. The methodaccording to claim 11, wherein the high risk HPV gene-product is encodedby the HPV E2 or the HPV E6 gene.
 15. The method according to claim 11,wherein the high risk HPV gene-product is encoded by the HPV L1 or theHPV L2 gene.
 16. A method of preparing a uterine cervical cytologysample for microscopic evaluation, said method comprising: (a) obtainingat least two slides having the uterine cervical cytology sampledeposited thereon; (b) staining the uterine cervical sample on at leastone of the slides by in situ hybridization for p16^(INK4a) mRNA with anagent that specifically binds p16^(INK4a) mRNA and generates a firstreporter signal; and (c) staining the uterine cervical sample on atleast one of the slides by in situ hybridization for a high risk HPV(hrHPV) mRNA with an agent that specifically binds the high risk HPVmRNA and generates a reporter signal, wherein the hrHPV is encoded by agene selected from the group consisting of HPV E7 gene, HPV E2 gene, HPVE6 gene, HPV L1 gene, and HPV L2 gene, wherein the stain for the hrHPVis an agent that generates a second reporter signal, wherein the firstreporter signal is distinguishable from the second reporter signal. 17.The method of claim 16, wherein the agent generating the first reportersignal and the agent generating the second reporter signal arefluorescent labels or colored compounds.
 18. The method according toclaim 16, wherein the high risk HPV gene-product is encoded by the HPVE7 gene.
 19. The method according to claim 16, wherein the high risk HPVgene-product is encoded by the HPV E2 or the HPV E6 gene.
 20. The methodaccording to claim 16, wherein the high risk HPV gene-product is encodedby the HPV L1 or the HPV L2 gene.